Chromatography of lipids using
a glass column filled with a suitable material is a common and useful method for
fractionation of lipid classes either on an analytical or a semi-preparative scale. While
this methodology leads to fractions which need to be later quantified, HPLC allows to
separate and quantify lipid compounds in a single process if a convenient detector is
present.

procedure which is based on the
importance of ionic groups present in some lipid molecules

1.Adsorption chromatography

This separation is based on differences in the
degree of adsorption of lipid components onto a solid and immobilized phase.

HISTORY

Curiously, this industrial and laboratory technique was discovered thanks to a botanist
who tried to analyze vegetal pigments which are also coloured lipids. MichaelTswett (1872-1920) is credited with developing and publishing the first
concept and technique of chromatography (column chromatography). Tswett was aware that his
results possessed significance beyond the mere separation of chlorophyll,
xanthophyll, and
carotene. The title of one of his papers was "On a new category of adsorption
phenomena and their application to biochemical analysis" (Tswett M, Ber Deut
Botan Ges 1906, 24, p.316 and 384; Chem Zeutr 1906, 72, 892 and 77, 1286; Chem Ber 1908,
41, 1352). The work of Tswett did not achieve at that time the recognition afforded
it today. At that time chemists were not attracted by micro-methods and Tswett was a
botanist.
A survey of the life and scientific acitvities of Tswett may be found in
LC-GC
north America.

Column chromatography was rediscovered and revived by Kuhn
in 1931 also in the course of studies on carotenoids (Kuhn R et al., Z Physiol
Chem 1931, 197, 141; Ber 1951, 64, 1349). This time this technique received wide
attention likely due to the general development of biochemistry which necessitated
refinement of the methods of separations. Despite the interest of lipid chemists in the
carotenoid substances, the development of chromatographic methods progressed more rapidly
with amino acids and carbohydrates than with lipids. Prior to 1950 only a few references
to chromatography of fatty substances can be found. In 1936, Thannhauser SJ described the
use of an aluminium oxide column to separate phospholipids (J Biol Chem 1936, 116, 527).
In 1948, Swain LA described the separation of unsaponifiable lipids into three
fractions
on an alumina column (Can Chem 1948, 32, 533). The use of silicic acid to
separate various lipids was described for the first time in 1952 by Borgstrom
B (Acta
Physiol Scand 1952, 25, 101).

The retention results in a variety of mechanisms including hydrogen bonding, Van der
Waals' forces and also ionic bonding. The solid phase is relatively polar (normal
chromatography) and the more polar the lipid, the more strongly is it adsorbed. Thus, the
lipids are eluted by increasingly polar solvents. This technique has a low resolution when
used at low pressure (Solid Phase Extraction or SPE) but has a high resolution (high
performance) when run at high pressure using a stationary phase made of fine particles
(HPLC). The former is restricted to the fractionation of complex mixtures into two or
three less complex ones, the later being adopted to analyze and quantify purified
fractions.

Various procedures were described according to the types of lipids separated and the
complexity of the sample studied.

The strategy to be adopted depends on the aim of the study, the available instruments and
on the second analysis step which follows the first fractionation step.

1-
When all the lipid classes must be studied (non polar and polar lipids), the following
procedures are proposed to the analyst: